Citation of documents: Please do not cite the URL that is displayed in your browser location input, instead use the DOI, URN or the persistent URL below, as we can guarantee their long-time accessibility.

Abstract

This thesis characterizes the extent of regulatory T cell–mediated immune suppression in breast cancer and explores the possibilities for its potential therapeutic modulation. Based on initial results obtained from a pilot clinical study of adoptive transfer of autologous bone marrow derived T cells in advanced metastatic breast cancer patients, we observed that T-cell unresponsiveness in 53% of study patients was associated with an existing immune suppression exerted by Treg. Further IFN-γ secretion assays with primary breast cancer samples revealed that T-cell activation, proliferation as well as CTL responses upon tumour antigen stimulation were hampered when Treg were present. Flow cytometric data displayed that in primary breast cancer patients the bone marrow (BM) contained significantly lower frequencies of Treg than the peripheral blood (PB) (p<0.001), which may at least partially explain the improved functional potential of BM-derived memory T cells. Different migratory capacities of Treg in the BM and the PB were observed regarding breast cancer profiled chemokine receptors CCR2, CCR4 and CCR5, which might be responsible for this scenario. By removing Treg through CD25 targeting, the pre-existing T-cell anti-tumour immunity was evoked remarkably in both BM and PB, especially in otherwise less responding PB, as determined by IFN-γ ELISpot assay with polyvalent tumour antigens (MCF7 and KS24.22) as well as with defined tumour associated antigens (polypeptides). This rescuing effect in the PB provides an opportunity of utilizing PB instead of limited BM as a source for adoptive T cell transfer immunotherapy. As a pioneer exploitation, a large-scale Treg-depletion in a leukapheresis sample obtained from a metastatic breast cancer patient was operated with CliniMACS system under clinical-grade conditions (GMP). A sufficient depletion of Treg, a high viability of isolated cells and a satisfying recovery of total cells were documented. To exploit immunotherapy regarding eliminating Treg, an oral metronomic cyclophosphamide (CTX) treatment (50 mg/day) was performed to selectively deplete Treg in vivo. This pilot clinical trial was applied on 13 advanced metastatic breast cancer patients for 3 months. Clinical responses was observed in 64% of study patients accompanied by a rapid and effective reduction of Treg on Day14 (p=0.005) and by an augmentation of spontaneous anti-tumour T-cell immunity on Day70. Last but not least, in this thesis we found that Treg existing in breast cancer patients exhibit breast tumour antigen specificity, which was evidenced indirectly and directly. MCF7- or KS24.22-specific IFN-γ secretion was abrogated when Treg were added back in ELISpot assay. The production of immunosuppressive cytokine IL-10 was increased only when Treg encountered breast tumour antigens measured by ELISA and intracellular staining experiments. By applying a novelly designed Treg Specificity Assay, we detected a broad spectrum of tumour antigens recognized by Treg in at least 47% of tested breast cancer patients with Mammaglobin A as the most frequent one (85%).